Tracing explosives in soil with transcriptional regulators of Pseudomonas putida evolved for responding to nitrotoluenes

Abstract

SummaryAlthough different biological approaches for detection of anti‐personnel mines and other unexploded ordnance (UXO) have been entertained, none of them has been rigorously documented thus far in the scientific literature. The industrial 2,4,6 trinitrotoluene (TNT) habitually employed in the manufacturing of mines is at all times tainted with a small but significant proportion of the more volatile 2,4 dinitrotoluene (2,4 DNT) and other nitroaromatic compounds. By using mutation‐prone PCR and DNA sequence shuffling we have evolved in vitro and selected in vivo variants of the effector recognition domain of the toluene‐responsive XylR regulator of the soil bacterium Pseudomonas putida that responds to mono‐, bi‐ and trinitro substituted toluenes. Re‐introduction of such variants in P. putida settled the transcriptional activity of the cognate promoters (Po and Pu) as a function of the presence of nitrotoluenes in the medium. When strains bearing transcriptional fusions to reporters with an optical output (luxAB, GFP) were spread on soil spotted with nitrotoluenes, the signal triggered by promoter activation allowed localization of the target compounds on the soil surface. Our data provide a proof of concept that non‐natural transcription factors evolved to respond to nitroaromatics can be engineered in soil bacteria and inoculated on a target site to pinpoint the presence of explosives. This approach thus opens new ways to tackle this gigantic humanitarian problem.

A standardized workflow for surveying recombinases expands bacterial genome‐editing capabilities

“…P. putida derives this sturdiness from its EDEMP cycle, a series of metabolic pathways that facilitate NAD(P)H production via carbon recycling processes (Nikel et al ., 2015). An adaptability to a myriad of harsh conditions has made P. putida a popular focus of biosynthetic studies aimed at industrial biotransformations as well as soil and water bioremediation (Garmendia et al ., 2008; Puchałka et al ., 2008; Poblete‐Castro et al ., 2012; Loeschcke and Thies, 2015). Unfortunately, the collection of genetic tools available in Pseudomonads is limited in scope and, when used in conjunction, is subject to bottlenecks that hinder the generation of a biotechnological chassis (Martínez‐García and de Lorenzo, 2011; Martínez‐García et al ., 2011, 2014).…”

Molecular Manipulations for Enhancing Luminescent Bioreporters Performance in the Detection of Toxic Chemicals

“…The same XylR regulator was employed to detect nitrotoluenes, predominant land mine components [21]. Two experimental strategies were employed to generate combinatorial XylR libraries, produced either by shuffling DNA segments of XylR with those of the homologous N-terminal domain of the phenol-responding regulator DmpR, or by random introduction of single amino-acid changes by errorprone PCR.…”

Systemic approaches to biodegradation

“…Also, molecular fingerprinting techniques based on DNA arrays are allowing a better characterization of the environment, allowing precise profiling of the local bacterial population and their variations in time, or in response to human interventions (Yin et al, 2007). An interesting approach is the use of in vitro evolved transcription regulators able to recognize novel compounds, which can potentially also be used to increase the metabolic potential of bacterial strains (Garmendia et al, 2008). Also, proteomic technologies are starting to be used for the enzymatic characterization of a specific environment (Zhao & Poh, 2008) However, despite having a strong potential, it should be noted that these approaches are only able to identify the presence of previously characterized enzymes and species, However, many of the bacterial strains that were isolated in the laboratory as good 'biodegraders' do not seem to play such an important role under natural conditions (Wackett, 2004b), and it will not be surprising that many yet unknown species contribute to biodegradation, particularly in view of the large microbial diversity (Curtis et al, 2002), for which it has been estimated that o 1% of the natural bacterial population can be cultured in the laboratory .…”